1. Field of the Invention
The present invention relates to human chemokine xcex2-6 agonist and antagonist polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques. The chemokine xcex2-6 antagonists of the present invention may be employed to treat rheumatoid arthritis, lung inflammation, allergy, asthma, infectious diseases and to prevent inflammation and atherosclerosis. The chemokine xcex2-6 agonists may be empolyed to myeloprotect patients undergoing chemotherapy. Chemokine xcex2-6 (Ckxcex2-6) is also refered to herein as MPIF-2 and eotaxin-2.
2. Related Art
There are three forms of monocyte chemotactic protein, namely, MCP-1, MCP-2 and MCP-3. All of these proteins have been structurally and functionally characterized and have also been cloned and expressed. MCP-1 and MCP-2 have the ability to attract leukocytes (monocytes, and leukocytes), while MCP-3 also attracts eosinophils and T lymphocytes (Dahinderi, E., et al, J. Exp. Med. 179:751-756 (1994)).
Initially, human monocyte-specific attracting factor, was purified from a glioma cell line and a monocytic cell line. Matsushima, K., et al, J. Exp. Med. 169:1485-1490 (1989). This factor was originally designated glioma-derived chemotactic factor (GDCF) and monocyte chemotactic and activating factor (MCAF) by Matsushima, et al. This factor is now referred to as MCP-1. Subsequent cloning of the cDNA for MCP-1 showed it to be highly similar to the murine JE gene. The JE gene could be massively induced in murine fibroblasts by platelet-derived growth factor. Cochran, B. H., et al, Cell 33:939-947 (1983). Murine JE is highly similar to MCP-1. The MCP-1 protein is 62% identical to murine JE in a region of 68 shared N-terminal residues. It is widely accepted that JE and MCP-1 are species homologs.
A method of suppressing tumor formation in a vertebrate by administering JE/MCP-1 has been disclosed in PCT application WO-92/20372, along with methods of treating localized complications of malignancies and methods of combatting parasitic infection by administering JE/MCP-1. Expression of the JE/MCP-1 protein in malignant cells was found to suppress the cells ability to form tumors in vivo.
Human MCP-1 is a basic peptide of 76 amino acids with a predicted molecular mass of 8,700 daltons. MCP-1 is inducibly expressed mainly in monocytes, endothelial cells and fibroblasts. Leonard, E. J. and Yoshimura, T., Immunol. Today 11:97-101 (1990). The factors which induce this expression is IL-1, TNF or lipopolysaccharide treatment.
Other properties of MCP-1 include the ability to strongly activate mature human basophils in a pertussis toxin-sensitive manner. MCP-1 is a cytokine capable of directly inducing histamine release by basophils, (Bischoff, S. C., et al., J. Exp. Med. 175:1271-1275 (1992)). Furthermore, MCP-1 promotes the formation of leukotriene C4 by basophils pretreated with Interleukin 3, Interleukin 5, or granulocyte/macrophage colony-stimulating factor. MCP-1 induced basophil mediator release may play an important role in allergic inflammation and other pathologies expressing MCP-1.
Clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) reveal the primary structure of the MCAF polypeptide to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. Furutani, Y. H., et al., Biochem. Biophys. Res. Commu. 159:249-55 (1989). The complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2) has also been determined. This peptide attracts human monocytes but not neutrophils. It was established that GDCF-2 comprises 76 amino acid residues. The peptide chain contains 4 half-cysteines, at positions 11, 12, 36 and 52, which create a pair of loops, clustered at the disulfide bridges. Further, the MCP-1 gene has been designated to human chromosome 17. Mehrabian, M. R., et al., Genomics 9:200-3 (1991).
Certain data suggests that a potential role for MCP-1 is mediating monocytic infiltration of the artery wall. Monocytes appear to be central to atherogenesis both as the progenitors of foam cells and as a potential source of growth factors mediating intimal hyperplasia Nelken, N. A., et al., J. Clin. Invest. 88:1121-7(1991). It has also been found that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with rheumatoid arthritis and that synovial tissue macrophages are the dominant source of this cytokine. MCP-1 levels were found to be significantly higher in synovial fluid from rheumatoid arthritis patients compared to synovial fluid from osteoarthritis patients or from patients with other arthritides. Koch, A. E., et al., J. Clin. Invest. 90:772-9 (1992).
MCP-2 and MCP-3 are classified in a subfamily of proinflammatory proteins and are functionally related to MCP-1 because they specifically attract monocytes, but not neutrophils. Van Damme, J., et al., J. Exp. Med. 176:59-65 (1992). MCP-3 shows-71% and 58% amino acid homology to MCP-1 and MCP-2 respectively. MCP-3 is an inflammatory cytokine that regulates macrophage functions.
The transplantation of hemolymphopoietic stem cells has been proposed in the treatment of cancer and hematological disorders. Many studies demonstrate that transplantation of hematopoietic stem cells harvested from the peripheral blood has advantages over the transplantation of marrow-derived stem cells. Due to the low number of circulating stem cells, there is a need for induction of pluripotent marrow stem cell mobilization into the peripheral blood. Reducing the amount of blood to be processed to obtain an adequate amount of stem cells would increase the use of autotransplantation procedures and eliminate the risk of graph versus host reaction connected with allotransplantation. Presently, blood mobilization of marrow CD34+ stem cells is obtained by the injection of a combination of agents, including antiblastic drugs and G-CSF or GM-CSF. Drugs which are capable of stem cell mobilization include IL-1, IL-7, IL-8, and NIP-1a Both IL-1 and IL-8 demonstrate proinflammatory activity that may be dangerous for good engrafting. IL-7 must be administered at high doses over a long duration and MIP-1a is not very active as a single agent and shows best activity when in combination with G-CSF.
In accordance with one aspect of the present invention, there is provided a novel full-length or mature polypeptide, as well as biologically active, diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding a polypeptide of the present invention including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the Ckxcex2-6 polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit No. 75703 on Mar. 10, 1994. The nucleotide sequence determined, at least in part, by sequencing the deposited Ckxcex2-6 clone, which is shown in FIG. 1 (SEQ ID NO: 1), contains an open reading frame encoding a polypeptide of 119 amino acid residues, with a leader of about 26 amino acid residues. The amino acid sequence of the mature Ckxcex2-6 protein is shown in FIG. 1, as amino acid residues 1-93 of SEQ ID NO:2.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding an Ckxcex2-6 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding an Ckxcex2-6 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2) excepting the N-terminal methionine; (c) a nucleotide sequence encoding an Ckxcex2-6 polypeptide having the amino acid sequence at positions 1-93 in FIG. 1 (SEQ ID NO-2); (d) a nucleotide sequence encoding the Ckxcex2-6 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75703; (e) a nucleotide sequence encoding the Ckxcex2-6 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75703 excepting the N-terminal methionine; (f) a nucleotide sequence encoding the mature Ckxcex2-6 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75703; and (g) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.
The present invention further relates to variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of the Ckxcex2-6 polypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or the polypeptides encoded by the cDNA of the deposited clone. The variants of the polynucleotides can be a naturally occurring allelic variant of the polynucleotides or a non-naturally ocurring variant of the polynucleotides.
Further embodiments of the invention include islolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% homologous or identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f) or (g), above. These polynucleotides which hybridize do not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
The invention further provides an isolated Ckxcex2-6 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the Ckxcex2-6 polypeptide having the complete amino acid sequence, including the leader sequence shown in FIG. 1 (SEQ ID NO:2); (b) the amino acid sequence of the Ckxcex2-6 polypeptide having the complete amino acid sequence, including the leader sequence shown in FIG. 1, excepting the N-terminal methionine; (c) the amino acid sequence of the mature Ckxcex2-6 polypeptide (without the leader) having the amino acid sequence at positions 1-93 in FIG. 1 (SEQ ID NO:2); (d) the amino acid sequence of the Ckxcex2-6 polypeptide having the complete amino acid sequence, including the leader sequence, encoded by the cDNA clone contained in ATCC Deposit No. 75703; (e) the amino acid sequence of the Ckxcex2-6 polypeptide having the complete amino acid sequence, including the leader sequence, encoded by the cDNA clone contained in ATCC Deposit No. 75703, excepting the N-terminal methionine; and (f) the amino acid sequence of the mature Ckxcex2-6 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75703.
Polypeptides of the present invention also include homologous polypeptides having an amino acid sequence with at least 90% identity, and more preferably at least 95% identity to those described in (a), (b), (c), (d), (e) or (f) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope bearing portion of an Ckxcex2-6 polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e) or (f) above. Peptides or polypeptides having the amino acid sequence of an epitope bearing portion of an Ckxcex2-6 polypeptide of the invention include portions of an Ckxcex2-6 polypeptide with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of an Ckxcex2-6 polypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or (f), above.
The present invention also provides, in another aspect, pharmaceutical compositions comprising an Ckxcex2-6 polynucleotide, probe, vector, host cell, polypeptide, fragment, variant, derivative, epitope bearing portion, antibody, antagonist or agonist.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, for stem cell mobilization, myeloprotection and neuronal protection, to treat tumors, to promote wound healing, to combat parasitic infection and to regulate hematopoiesis.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of Ckxcex2-6 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated Ckxcex2-6 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of Ckxcex2-6 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an Ckxcex2-6 antagonist. Preferred antagonists for use in the present invention are Ckxcex2-6 specific or CCR3 receptor specific antibodies.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides. In another embodiment, the invention provides an isolated antibody that binds specifically to an Ckxcex2-6 polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e) or (f) above.
The invention further provides methods for isolating antibodies that bind specifically to an Ckxcex2-6 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
In accordance with another aspect of the present invention, there are provided agonists which mimic the polypeptide of the present invention and bind to receptors to elicit second messenger responses.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of rheumatoid arthritis, lung inflammation, histamine-mediated allergic reactions, infectious diseases, hyper-eosinophilic syndromes, silicosis, sarcoidosis and to prevent auto immune and chronic inflammation and atherosclerosis. Alternatively, such polypetides can be used to inhibit production of IL-1 and TNFxcex1, to treat aplastic anemia, myelodysplastic syndrome, asthma and arthritis.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases relating to underexpression or overexpression of the polypeptides and for detecting susceptibility to diseases related to mutations in the nucleic acid sequences encoding a polypeptide of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, for example, synthesis of DNA and manufacture of DNA vectors, for the purpose of developing therapeutics and diagnostics for the treatment of human disease.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by an Ckxcex2-6 polypetide, which involves contacting cells which express the Ckxcex2-6 polypeptide with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
For a number of disorders, it is believed that significantly higher or lower levels of Ckxcex2-6 gene expression can be detected in certain tissues or bodily fluids (e.g., serum, plama, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a standard Ckxcex2-6 gene expression level; i.e., the Ckxcex2-6 expression level in tissue or bodily fluids from an individual not having the disorder, which involves: (a) assaying the Ckxcex2-6 gene expression level in cells or body fluid of an individual; (b) comparing the Ckxcex2-6 gene expression level with a standard Ckxcex2-6 gene expression level, whereby an increase or decrease in the assayed Ckxcex2-6 gene expression level compared to the standard expression level is indicative of a disorder. Such disorders include leukemia, chronic inflammation, autoimmune diseases, and solid tumors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.